Molecular conjugates for use in treatment of cancer

ABSTRACT

A molecular conjugate is provided having the formula: 
                         
wherein R 1  is a de-hydroxyl or de-amino moiety respectively of a hydroxyl-bearing or amino-bearing biologically active molecule or an analog or derivative thereof, and Z is —O— or —NH—, respectively, Y is a straight or branched alkyl having 1 to 20 carbons that may be optionally substituted with one or more phenyl, a cycloalkyl optionally substituted with one or more alkyl or phenyl, or an aromatic group optionally substituted with one or more alkyl groups, electron-withdrawing groups, or electron-donating groups; and R 2  is —CH═CH(W), —CH(OH)CH(OH)W, or —C(O)H, where W can be H, a straight or branched alkyl having 1 to 20 carbons that may be optionally substituted with one or more phenyl, a cycloalkyl optionally substituted with one or more alkyl or phenyl, or an aromatic group optionally substituted with one or more alkyl groups, electron-withdrawing groups, or electron-donating groups.

FIELD OF THE INVENTION

The present invention generally relates to chemical compounds andmethods for use in treating patients. More particularly, the presentinvention is directed to molecular conjugates for use in cancertreatment. Specifically, the present invention relates toTransferrin-drug conjugates, methods and intermediates useful in theformation thereof, and methods for treating a patient therewith.

BACKGROUND OF THE INVENTION

A number of anti-cancer drugs are currently in clinical use for thetreatment of various cancers. For example, paclitaxel and taxotere aretwo promising anti-cancer drugs used to treat breast and ovariancancers, and which hold promise for the treatment of other cancers suchas skin, lung, head and neck carcinomas. Other promisingchemotherapeutic agents are being developed or tested for treatment ofthese and other cancers. Compounds such as paclitaxel, taxotere, andother taxanes, camptothecins, epothilones and quassinoids, as well asother compounds exhibiting efficacy in cancer treatment, are ofconsiderable interest. Of special interest are natural product drugswith demonstrated anticancer activity, in vitro and in vivo. Suchcompounds are desirable, for example, for their potential availabilityfrom renewable resources.

However, many identified anti-cancer compounds present a number ofdifficulties with their use in chemotherapeutic regimens. One particularproblem relates to the aqueous insolubility of many anti-cancercompounds, which creates significant problems in developing suitablepharmaceutical formulations useful for chemotherapy. In an attempt toincrease the aqueous solubility of these drugs, they are oftenformulated with various carrier compounds. However, these carriercompounds often cause various adverse side effects in a patient treatedwith the formulation. For example, paclitaxel and camptothecin and theiranalogs are generally formulated with a mixture of polyethoxylatedcastor oil (Cremophore) and ethanol. This mixture has been reported tocause side effects in clinical trials, which include neutropenia,mucositis, cardiac and neurological toxicities, hypersensitivity,histamine release and severe allergic reactions.

Another problem with the use of such chemotherapeutic agents in cancertreatment is the difficulty targeting cancer cells without adverselyaffecting normal, healthy cells. For example, paclitaxel exerts itsantitumor activity by interrupting mitosis and the cell divisionprocess, which occurs more frequently in cancer cells than in normalcells. Nonetheless, a patient undergoing chemotherapy treatment mayexperience various adverse side effects associated with the interruptionof mitosis in normal, healthy cells.

Accordingly, it would be highly desirable to develop chemical compoundsand methods for use in directly targeting cancer cells withchemotherapeutic agents in cancer treatment regimens. This, in turn,could lead to reduction or elimination of toxic side effects fromcarrier compounds, more efficient delivery of the drug to the targetedsite, and reduction in dosage of the administered drug and a resultingdecrease in toxicity to healthy cells and in the cost of administeringthe chemotherapeutic regimen.

One particular approach of interest is the use of anti-cancer drugmoieties that have been conjugated to tumor-recognizing molecules. Forexample, U.S. Pat. No. 6,191,290 to Safavy discusses the formation anduse of a taxane moiety conjugated to a receptor ligand peptide capableof binding to tumor cell surface receptors. Safavy in particularindicates that such receptor ligand peptides might be BBN/GRPreceptor-recognizing peptide, a somatostatin receptor-recognizingpeptide, an epidermal growth factor receptor-recognizing peptide, amonoclonal antibody or a receptor-recognizing carbohydrate.

One tumor-recognizing molecule of particular interest is the humanprotein Transferrin. Transferrin is a serum glycoprotein ofapproximately 79550 molecular weight, which is involved in irontransport to developing red cells for hemoglobin synthesis. It has avery high binding affinity for ferric iron so that essentially no freeferric iron, a very toxic form of iron, occurs in plasma. Further, theiron requirement of growing cells is provided by diferric Transferrin(each protein molecule specifically binds with two Fe³⁺ ions to formsalmon-pink complexes) which binds to receptors on the cell membraneleading to an internalization of the Transferrin-receptor complex whichthen leads to a release of iron to the cytoplasm of the cell and returnof the apoTransferrin-receptor complex to the cell surface and releaseof the apoTransferrin from the receptor. It has been demonstrated thatgrowing cells have Transferrin receptors on their cell surface whereasstatic cells either do not or have very low numbers of Transferrinreceptors. Further, cancer cells have been demonstrated to have a highnumber of Transferrin receptors and interestingly, drug resistant cancercells have an even greater number of Transferrin receptors. The presenceof Transferrin receptors on cancer cells but not on normal cellssuggests that Transferrin conjugates could provide a selective way oftargeting agents to cancer cells. For instance, as reported by Yeh etal., “Killing of Human Tumor Cells in Culture with Adriamycin Conjugatesof Human Transferrin”, Clin. Immunol. Immunopathol. 32, 1–11 (1984), andby Sizensky et al., “Characterization of the Anti-Cancer Activity ofTransferrin-Adriamycin Conjugates”, Am. J. Reprod. Immunol. 27:163–166(1992), Transferrin-adriamycin conjugates have a higher therapeuticindex than free adriamycin for cancer therapy.

Other works suggest a promising approach to cancer therapies utilizingTransferrin conjugated with various chemotherapeutic drugs, such asDoxorubicin (Kratz et al., “Transferrin conjugates of Docorubicin:Synthesis, Characterization, Cellular Uptake, and in Vitro Efficacy”, J.Pharm Sci., 87, 338–346 (1998)) and Mytomycin C (Tanaka et al.,“Synthesis of Transferrin-Mitomycin C Conjugate as a Receptor-MediatedDrug Targeting System”, Biol. Pharm. Bull. 19, 774–777 (1996)).

An attempt at an effective Transferrin-paclitaxel conjugate was reportedby Bicamumpaka et al., “In Vitro Cytotoxicity of Paclitaxel-TransferrinConjugate on H69 Cells”, Oncol. Rep., 5, 1381–1383 (1998). Inparticular, Bicamumpaka et al. synthesized 2′-glutaryl-hexanediaminepaclitaxel, which was then coupled to Transferrin using a glutaraldehydelinker through an amino of the 2′-glutaryl-hexanediamine group. However,Bicamumpaka reported that the capacity of the resultingTransferrin-paclitaxel conjugate to inhibit growth of H69 cells was 5.4times less than that of the native paclitaxel drug.

Accordingly, it can be seen that there is a need to provide new chemicalcompounds for linking chemotherapeutic agents to various molecules, suchas Transferrin, the receptor ligand peptides recognized by Safavy, orother proteins, antibodies, lectins or other substances that may becomeattached to the surface of a cell. There is also a need to providemethods for forming such compounds. It can further be seen that there isa need for new molecular conjugates for use in treating cancer, andTransferrin-drug conjugates in particular. Finally, there is a need fornew methods of administering chemotherapeutic pharmaceuticalformulations to patients for use in cancer treatment regimens, such asthrough the use of improved molecular conjugates such asTransferrin-drug conjugates. The present invention is directed tomeeting these needs.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide new and usefulcompositions of molecular conjugates of hydroxyl-bearing oramino-bearing drugs.

It is a further object to provide compositions of Transferrin-drugconjugates for use in treating cancer.

It is another object to provide intermediate compounds for use informing molecular conjugates, such as Transferrin-drug conjugates, foruse in treating cancer.

It is yet another object to provide efficient methods for the formationof molecular conjugates, and Transferrin-drug conjugates in particular.

A still further object is to provide new and useful methods foradministering chemotherapeutic agents to patients that reduce oreliminate side effects conventionally experienced by cancer patients.

A still further object of the present invention is to provide methodsfor efficiently concentrating chemotherapeutic agents in cancer cells ofa patient.

According to the present invention, then, compounds useful in theformation of molecular conjugates, such as Transferrin-drug conjugates,are provided. The compounds have the generalized formula:

wherein R₁ is a de-hydroxyl or de-amino moiety respectively of ahydroxyl-bearing or amino-bearing biologically active molecule or ananalog or derivative thereof, and Z is —O— or —NH—, respectively, Y is astraight or branched alkyl having 1 to 20 carbons that may be optionallysubstituted with one or more phenyl, a cycloalkyl optionally substitutedwith one or more alkyl or phenyl, or an aromatic group optionallysubstituted with one or more alkyl groups, electron-withdrawing groups,or electron-donating groups; and R₂ is —CH═CH(W), —CH(OH)CH(OH)W, or—C(O)H, where W can be H, a straight or branched alkyl having 1 to 20carbons that may be optionally substituted with one or more phenyl, acycloalkyl optionally substituted with one or more alkyl or phenyl, oran aromatic group optionally substituted with one or more alkyl groups,electron-withdrawing groups, or electron-donating groups.

R₁ is preferably a biologically active molecule that is useful in cancertherapy, which may further be a cancer therapeutic drug such as taxanes,camptothecins, epothilones, cucurbitacins, quassinoids, anthracyclines,and their analogs and derivatives. R₁ can specifically be a moietyselected from the group consisting of 7-dehydroxyl paclitaxel,10-dehydroxyl paclitaxel, 2′-dehydroxyl paclitaxel, 3′-de-benzamidopaclitaxel, 3-dehydroxyl cholesterol and 20-dehydroxyl camptothecin, andtheir analogs and derivatives.

These and other objects of the present invention will become morereadily appreciated and understood from a consideration of the followingdetailed description of the exemplary embodiments of the presentinvention when taken together with the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chemical reaction scheme for forming a 7-acyl-pentanalpaclitaxel linker compound for use in forming a Transferrin-7-paclitaxelconjugate;

FIG. 2 shows a chemical reaction scheme for forming aTransferrin-3-cholesterol conjugate by linking a 3-acyl-pentanalcholesterol linker compound with Transferrin;

FIG. 3 shows a chemical reaction scheme for forming aTransferrin-20-camptothecin conjugate by linking a 20-acyl-pentanalcamptothecin linker compound with Transferrin;

FIG. 4( a) shows a chemical reaction scheme for forming a2′-acyl-pentanal paclitaxel linker compound for use in forming aTransferrin-2′-paclitaxel conjugate;

FIG. 4( b) shows a chemical reaction scheme for forming a2′-acyl-hexanal paclitaxel linker compound for use in forming aTransferrin-2′-paclitaxel conjugate;

FIG. 4( c) shows a chemical reaction scheme for forming a2′-acyl-nonanal paclitaxel linker compound for use in forming aTransferrin-2′-paclitaxel conjugate;

FIGS. 5–7 are graphs demonstrating the cytotoxicity against KB cells ofa Transferrin-3-cholesterol conjugate, a Transferrin-rhodamine123conjugate and a Transferrin-7-paclitaxel conjugate, respectively, atvarious concentrations under Protocol A;

FIGS. 8–10 are graphs demonstrating the cytotoxicity against Lu cells ofa Transferrin-3-cholesterol conjugate, a Transferrin-rhodamine123conjugate and a Transferrin-7-paclitaxel conjugate, respectively, atvarious concentrations under Protocol A;

FIGS. 11–13 are graphs demonstrating the cytotoxicity against hTERTcells of a Transferrin-3-cholesterol conjugate, aTransferrin-rhodamine123 conjugate and a Transferrin-7-paclitaxelconjugate, respectively, at various concentrations under Protocol A;

FIGS. 14–16 are graphs demonstrating the cytotoxicity against KB, Lu andhTERT cells, respectively, of a Transferrin-7-paclitaxel conjugate atvarious concentrations under Protocol A; and

FIGS. 17–19 are graphs demonstrating the cytotoxicity against KB, Lu andhTERT cells, respectively, of a Transferrin-7-paclitaxel conjugate atvarious concentrations under Protocol B.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The present invention provides new molecular conjugates, and inparticular new Transferrin-drug conjugates for use in treating cancer ina patient. Additionally, the present invention is directed to novelintermediate compounds for use in linking biologically active moleculesto carrier molecules such as Transferrin or other molecules. Inparticular, the present invention provides aldehyde ester and amidoderivatives, respectively, of hydroxyl-bearing and amine-bearingbiologically active molecules, such as cancer therapeutic drugs andanalogs and derivatives thereof, as well as precursors thereto, whichcan be linked to carrier molecules such as human Transferrin proteinthrough the formation of Schiff bases between the aldehyde functionalityof the ester or amide linkage and various amino functionalities of theTransferrin molecule or other protein.

The present invention also provides an efficient protocol for thesynthesis of Transferrin conjugates, or other molecular conjugates, ofvarious hydroxyl-bearing or amino-bearing biologically active compounds,and intermediates thereto. A generalized process includes coupling suchhydroxyl-bearing or amino-bearing biomolecules with an appropriateacylating agent, such as a carboxylic acid or acid halide, having adouble bond, preferably a terminal olefin. A rapid and highly efficientoxidation of the terminal olefin site using catalytic osmium tetroxidefollowed by cleavage of the resulting diol to aldehyde provides asuitable precursor for synthesis of Transferrin conjugates or othermolecular conjugates. The final step in the synthetic sequence of theseadducts is the treatment of the aldehyde with a carrier molecule such asthe blood protein Transferrin to make biomolecules attached to monomericTransferrin, which are found to have an increased biological activity.In place of Transferrin, the present invention broadly contemplates thatcarrier molecules may include any molecule having at least oneaccessible amino functionality through which a Schiff base may be formedwith the aldehyde functionality of the ester and amido linker compoundsof biologically active molecules, as disclosed herein.

It should also be appreciated that the present invention broadlyconstrues the term “biologically active molecule” as including anymolecule that generally affects or is involved in or with one or morebiological processes in cells, tissues, vessels, or the like. Suchbiologically active molecules may comprise drugs, antibodies, antigens,lectins, dyes, stains, tracers or any other such molecule. Inparticular, hydroxyl-bearing or amino-bearing molecules contemplated foruse in the invention include paclitaxel, docetaxel and other taxanes,cholesterol, rhodamine 123, camptothecins, epothilones such asepothilone B, cucurbitacins, quassinoids such as glaucarubolone,brusatol and bruceantin, anthracyclines such as adriamycin, daunorubicinand the like, and their analogs and derivatives, as well as othercompounds. The term “molecular conjugate” should be understood tobroadly encompass any compound comprising a biologically active moleculelinked to a carrier molecule according to the present invention, such asthrough the ester and amide Schiff base linkages disclosed herein.

It should further be understood that, while the focus of this work isdirected to cancer therapy, the present application contemplates theconjugation according to the present invention of various proteins orother carrier molecules with biologically active molecules directedtoward other applications.

I. Transferrin-7-Paclitaxel Conjugate

A Transferrin-7-paclitaxel conjugate can be formed according to thepresent invention. As shown in FIG. 1, paclitaxel is first converted toa 7-paclitaxel aldehyde ester through various intermediate compounds.The aldehyde ester is then linked to Transferrin to form aTransferrin-7-paclitaxel conjugate.

A. Preparation of 2′TBDMS paclitaxel:

Paclitaxel was first protected at the 2′-hydroxyl with TBDMS to form2′-TBDMS paclitaxel. While TBDMS is shown in the exemplary reaction, itshould be appreciated that other protecting groups, such as TROC, BOM,CBZ, benzyl, TES, EE or the like, may be used in place of TBDMS.

This material was prepared according to the procedures described by ProfGunda Georg et al in Tetrahedron Letters, vol 35, p 8931–8934, 1994 andcharacterized accordingly.

To a solution of paclitaxel (20.0 g, 23.45 mM) in dimethylformamide (150mL) was added imidazole (23.95 g, 351.7 mM) under nitrogen atmosphere,followed by the addition of TBDMSCI (49.5 g, 328.3 mM). The resultingsolution was stirred at ambient temperatures for 16 h under nitrogenatmosphere. The TLC examination at this stage confirmed completeconsumption of the starting material and the reaction was worked up byadding water (200 mL) and ethyl acetate (200 mL). The organic layer wasseparated and washed with water (2×100 mL), brine (50 mL) and dried overmagnesium sulfate, and thereafter filtered and evaporated to a residue,dried in a vacuum oven and used for the following reaction with nofurther purification.

B. Preparation of 7-hexenoate of 2′protected paclitaxel:

Next, the 7-hexenoate of the 2′-protected paclitaxel was formed byreaction with an acid preferably having terminal olefin. While5-hexenoic acid is used in the examples herein, it should be appreciatedthat the present invention contemplates other appropriate acylatingagents preferably having terminal olefin, although olefinic acylatingagents having the double bond further displaced from the end of thechain are also contemplated. For example, the present inventioncontemplates the use of acids of the formula:

or acid halides of the formula:

wherein X is a halogen and Y can be a straight or branched alkyl having1 to 20 carbons optionally substituted with one or more phenyl, acycloalkyl optionally substituted with one or more alkyl or phenyl, oran aromatic group optionally substituted with one or more alkyl orelectron-withdrawing or electron-donating groups. W can be H, a straightor branched alkyl having 1 to 20 carbons optionally substituted with oneor more phenyl, a cycloalkyl optionally substituted with one or morealkyl or phenyl, or an aromatic group optionally substituted with one ormore alkyl or electron-withdrawing or electron-donating groups.

Here, to a solution of 2′TBDMS paclitaxel (2.0 g, 2.07 mM) in methylenechloride (30 mL) was added 5-hexenoic acid (0.49 mL, 4.14 mM) followedby DIPC (0.81 mL, 5.18 mM) and 4-PP (0.095 g, 0.64 mM) under nitrogenatmosphere. The resulting reaction mixture was stirred for 4 h anddeemed complete by TLC analysis. The mixture was worked up by addingwater (50 mL) and ethyl acetate (90 mL), and the separated organic layerwas washed with water (50 mL), brine (50 mL) and dried over magnesiumsulfate. The resulting product was filtered and the solvent evaporatedto leave a residue which was subjected to the next reaction with nopurification.

C. Preparation of 7-aldehyde derivative of 2′ protected paclitaxel:

Oxidation of the terminal olefin site to the resulting diol, followed bycleavage of the terminal carbon provides a 7-aldehyde 2′-protectedderivative of paclitaxel. Where an acylating agent is used that has thedouble bond shifted from the end of the chain as discussed above (i.e.where W is not hydrogen in the above formula), it should be appreciatedthat cleavage of the double bond during this reaction removes theportion of the chain beyond the double bond. Also, while shown as asingle step in the exemplary processes, it should be appreciated thatthe diol of the formula:

(and corresponding diols of compounds described herein) may be isolatedby performing this step in an absence of NalO₄. Oxidative cleavage ofthe diol on treatment with NalO₄ provides the terminal aldehyde.

To a solution of 7-hexenoyl, 2′TBDMS paclitaxel (2.2 g, 2.07 mM) in ACNand THF (20 mL each) was added water (20 mL) followed by the addition ofNMO (0.49 g, 4.14 mM), NalO₄ (0.89 g, 4.14 mM) and OsO₄ solution int-BuOH (13.15 mg, 0.052 mM) under nitrogen atmosphere. The resultingreaction mixture was stirred for 2 h at ambient temperatures and wasworked up by adding ethyl acetate and water (100 mL each). The separatedorganic layer was washed with brine (20 mL) and filtered throughmagnesium sulfate and sodium hydrosulfite. The filtrate was evaporatedto dryness and subjected to desilylation reaction with no purificationof the crude product.

D. Preparation of the 7 aldehyde derivative of paclitaxel:

The 2′-position is deprotected as follows. To a solution of 7-aldehydederivative of 2′protected paclitaxel (2.25 g, 2.07 mM) in THF (50 mL)was added TBAF in THF (3.11 mL of 1.0 M solution, 3.11 mM) undernitrogen atmosphere at ambient temperatures. The resulting reactionmixture was stirred for 2 h and the TLC examination at this time showedno starting material. The mixture was worked up by adding ethyl acetate(200 mL) and 0.5N HCl (100 mL), and the separated organic layer waswashed with water (200 mL), brine (100 mL), and dried over magnesiumsulfate. The organic layer was filtered and evaporated to dryness,followed by purification on column chromatography using ethyl acetateand heptane to provide pure material in 60% overall yield. The compoundwas characterized by MS and ¹H NMR.

E. Preparation of Transferrin-7-Paclitaxel Conjugate

The aldehyde ester derivative may next be linked with Transferrin toform a Transferrin-7-paclitaxel conjugate having a conjugation number n(the number of paclitaxel molecules per Transferrin molecule), which wasfound to be 3, although it is contemplated that varying conditions mightproduce a Transferrin-7-paclitaxel conjugate having a conjugation numbern between 1 and 5. The present invention contemplates, of course, thatcarrier molecules such as other proteins having accessible aminofunctionalities may be used in place of Transferrin, and the conjugationnumber of the resulting molecular conjugate may vary accordingly.

Here, 2 ml of 19.04 mg (20 μmol) 7-acyl-pentanal paclitaxel in DMSO wasadded dropwise to 80 mg (1 μmol) Transferrin in PBS-buffer/DMSOsolution. Transferrin PBS-buffer/DMSO solution was prepared bydissolving Transferrin in 4 ml PBS (50 mmol pH 8.0), and 2 ml DMSO wasadded to Transferrin PBS solution at 0° C. The reaction mixture wasshaken by C24 incubator shaker (New Brunswick Scientific classic series,Edison, U.S.A.) at 37° C. for 8 h. The reaction mixture was filtered by5.0 μm filter unit. The clear filtrate was purified using FPLC on asuperdex HR200 column (2.0×30 cm) at 0.5 ml/min of 20 mM Tris-HCl (pH8.0). The fraction corresponding to Transferrin was collected and driedby lyophilization.

While not exemplified herein, it should be appreciated that molecularconjugates of 10-deacetyl paclitaxel are formed similarly to7-paclitaxel conjugates. For example, a Transferrin-10-acyl-hexanalpaclitaxel conjugate is formed similarly to the above-described processusing 10-deacetyl paclitaxel of formula:

as a starting compound and using 6-heptenoic acid as an acylating agent.Appropriate protections and deprotections of the 2′ and 7 positions maybe utilized as known in the art.II. Transferrin-3-Cholesterol Conjugate

A Transferrin-3-cholesterol conjugate was prepared to investigate theresults of linking a non-cytotoxic molecule to Transferrin. As discussedbelow, these results suggest that the Transferrin-drug conjugatesaccording to the present invention for use in treating cancer should beformed with cancer therapeutic agents having demonstrated in vitro or invivo cytotoxic activity. Various 3-cholesterol conjugates with otherproteins might be similarly prepared for comparison with the conjugationof such proteins with other biologically active compounds, such as forthe investigation of applications beyond those of cancer therapeuticmolecules, for example.

As shown in FIG. 2, cholesterol is first converted through variousintermediate compounds to a 3-cholesterol aldehyde ester, which is thenlinked to Transferrin to form a Transferrin-3-cholesterol conjugate. Aspreviously discussed, various other acylating agents as described abovemay be substituted for the 5-hexenoic acid used in the examples herein.

A. Preparation of 3-hexenoyl cholesterol:

To a solution of cholesterol (2.0 g, 5.17 mM) in methylene chloride (20mL) was added 5-hexenoic acid (0.68 mL, 5.69 mM) followed by theaddition of DCC (1.60 g, 7.76 mM) and 4-PP (0.115 g, 0.78 mM) undernitrogen atmosphere. The resulting reaction mixture was stirred atambient temperature for 1 h and worked up with the addition of methylt-butyl ether (60 mL). The urea was filtered off, and the product wastransferred to a separatory funnel, washed with 1N HCl (10 mL), water(30 mL) and brine (30 mL). The product was filtered after drying overMgSO₄ and the solvent was evaporated to leave a residue. The productobtained in >95% yield was characterized by ¹H NMR.

B. Preparation of 3-acyl-pentanal ester of cholesterol:

To a solution of 3-hexenoate of cholesterol (2.0 g, 4.14 mM) in THF andt-BuOH (10 mL each) was added water (5 mL) followed by the addition ofNMO (0.97 g, 8.3 mM), NalO₄ (1.78 g, 8.3 mM) and OsO₄ solution in t-BuOH(21.3 mg, 0.083 mM) under nitrogen atmosphere. The resulting reactionmixture was stirred for 16 h until complete conversion was observed byTLC analysis. Diatomaceous earth (1.6 g) was added to the reactionmixture and filtered. The filter cake was washed with ethyl acetate (100mL), and the filtrate was transferred to a separatory funnel and washedwith 1N HCl (15 mL), water (25 mL) and brine (15 mL) followed by dryingover MgSO₄. The filtered solution was evaporated to dryness followed bypurification on column chromatography to provide the product in 85%yield. The product was characterized by ¹H NMR. The NMR analysisrevealed that the internal double bond in the cholesterol system wasintact for the oxidation conditions.

C. Preparation of Transferrin-3-Cholesterol Conjugate

The aldehyde ester derivative may next be linked with Transferrin toform a Transferrin-3-cholesterol conjugate having a conjugation numbern.

1 ml of 1.21 mg (2.5 μmol) 3-acyl-pentanal cholesterol in DMSO was addeddropwise to 2 ml of 20 mg (0.5 μmol) Transferrin in PBS-buffer (50 mmolpH 7.0). The reaction mixture was shaken by C24 incubator shaker (NewBrunswick Scientific classic series, Edison, U.S.A.) at 37° C. for 30min. To this was added 0.5 ml of 1.527 mg (25 mmol) ethanolamine PBSsolution as a quenching agent to quench the reaction. The turbid mixturewas then centrifuged at 1000 g for 10 min at 4° C. and the clearsupernatant was purified using FPLC on a superdex HR200 column (2.0×30cm) at 0.5 ml/min of 20 mM Tris-HCl (pH 8.0). The fraction correspondingto Transferrin was collected and dried by lyophilization.

III. Transferrin-20-Camptothecin Conjugate

As shown in FIG. 3, camptothecin is first converted through variousintermediate compounds to a 20-camptothecin aldehyde ester, which isthen linked to Transferrin to form a Transferrin-20-camptothecinconjugate. The present invention contemplates that carrier moleculessuch as other proteins having accessible amino functionalities may besubstituted for Transferrin, and other acylating agents as describedabove may be substituted for 5-hexenoic acid.

A. Preparation of 20-hexenoyl camptothecin:

To a mixture of camptothecin (2.0 g, 5.74 mM) and 5-hexenoic acid (0.75mL, 6.31 mM) in DMF (40 mL) were added DIPC (0.99 mL, 6.31 mM) and 4-PP(0.13 g, 0.86 mM) under nitrogen atmosphere. The resulting reactionmixture was stirred for a period of 24 h at ambient temperatures. Afterconfirmation of complete consumption of camptothecin by TLC analysis,the reaction mixture was transferred to a separatory funnel usingmethylene chloride and water (200 mL each). The separated organic layerwas washed with brine and dried over MgSO₄, filtered and evaporated todryness. The solid residue was crystallized using methylene chloride andethyl acetate. Crystals were filtered to provide >98% pure material in70% yield and was characterized by MS and ¹H NMR data.

B. Preparation of 20-acyl-pentanal ester of camptothecin:

To a solution of 20-hexenoate camptothecin (1.0 g, 2.25 mM) in THF,Acetone and ACN (15 mL each) was added water (15 mL), followed by theaddition of NMO (0.53 g, 4.5 mM), NalO₄ (0.96 g, 4.5 mM) and OsO₄ (15.3mg, 0.06 mM) solution in t-BuOH under nitrogen atmosphere. The resultingreaction mixture was stirred at ambient temperatures for 4 h to completeconversion of the hexenoate to the pentanal derivative of camptothecin.The reaction mixture was partitioned between methylene chloride andwater (200 mL each). The organic layer was separated and washed with 1NHCl (10 mL), water (50 mL), brine (50 mL) and dried over MgSO₄, filteredand evaporated to dryness. The residue was purified by columnchromatography and the purified material was characterized by MS and ¹HNMR data.

C. Preparation of Transferrin-20-Camptothecin Conjugate

The aldehyde ester derivative may next be linked with Transferrin toform a Transferrin-20-camptothecin conjugate having a conjugation numbern.

The method for forming the Transferrin-20-camptothecin conjugate issimilar to the methods for forming the Transferrin-7-paclitaxelconjugate and Transferrin-3-cholesterol conjugate, as described above,which utilize DMSO and Transferrin PBS solution. Mass spectrometryanalysis of the resulting Transferrin-20-camptothecin conjugateindicated a coupling ratio of three camptothecin per Transferrinmolecule (i.e. n=3). The Circular Dichroism (CD) spectra of Transferrinand of the Transferrin-20-camptothecin conjugate were different,indicating that they might have changed overall conformation.

IV. Transferrin-Rhodamine123 Conjugate

A Transferrin-rhodamine123 conjugate was prepared to utilize thefluorescent properties of rhodamine123 for detection and visualization.The Transferrin-rhodamine 123 conjugate was prepared in a single stepusing glutaraldehyde as a linker between the free amino groups ofrhodamine123 and Transferrin, respectively. An activated aldehydecompound of the formula:

is believed to be formed as an intermediate in this reaction.I. Preparation of Transferrin-Rhodamine123 Conjugate

1 ml of 40 mg (0.5 μmol) Transferrin in Hepes-buffer saline (HBS, 150mmol NaCl, 10 mmol/L Hepes, pH 7.4) was mixed with 1 ml of 5 μmolrhodamine123 by vortexing for 4 min. 1 ml Glutaraldehyde (12.5 mmol inHBS) was added dropwise while vortexing for 4 min at room temperature.The coupling procedure was quenched by adding 0.5 ml of 25 mmolethanolamine HBS solution as a quenching agent and vortexing for 4 min.The mixture was transferred into dialysis tubing and dialyzed against 1L of HBS in the dark at 4° C. for 8 h. The turbid mixture was thencentrifuged at 1000 g for 10 min at 4° C. and the clear supernatantchromatographed at 0.5 ml/min of 20 mM Tris-HCl (pH 8.0) on a superdexHR200 column (2.0×30 cm). The fraction corresponding to Transferrin wascollected and dried by lyophilization.

V. 2′-Paclitaxel Compounds

As shown in FIGS. 4( a), 4(b) and 4(c), various 2′-paclitaxelintermediate compounds were formed, although formation of aTransferrin-2′-paclitaxel conjugate was problematic with smalleralkyl-chain aldehyde derivatives (such as using 5-hexenoic acid in theprocess as in FIG. 4( a)), presumably due to hindrance from thepositioning of the 2′-site in the concave region around C-13 of thehemispherical taxane skeleton. Accordingly, this result suggested that alonger alkyl-chain aldehyde derivative, such as one formed using alinking compound having a longer chain than does hexenoic acid, might beutilized to form a Transferrin-2′-paclitaxel conjugate according to thepresent invention. Further experimentation showed that 2′-heptanal and2′-nonanal Transferrin conjugates were more readily formed using asimilar chemical process, as shown in FIGS. 4( b) and 4(c),respectively. It should be noted that oleic acid (an eighteen carbonchain acid having a double bond at 9,10) was used for the formation ofthe 2′-nonanal paclitaxel compounds, as shown in FIG. 4( c).Additionally, given that Transferrin is a relatively large protein, thisresult also suggests that smaller carrier molecules, such as thoseproteins identified by Safavy, above, may be less hindered by theconcave structure of paclitaxel and could conjugate more readily withshorter 2′-paclitaxel alkyl-chain aldehyde derivatives, such as oneswhere Y<4 in the acylating agent formulas above.

A. Preparation of 2′hexenoate of paclitaxel:

To a solution of paclitaxel (2.0 g, 2.34 mM) and 5-hexenoic acid (0.31mL, 2.58 mM) in methylene chloride (25 mL) were added DCC (0.72 g, 3.51mM) followed by 4-PP (0.17 g, 0.5 mM) at 0° C. under nitrogenatmosphere. The resulting reaction mixture was stirred for 2 h, duringwhich time the reaction mixture was brought to ambient temperatures. Thereaction was monitored by TLC which confirmed completion of the reactionafter 2 h. The mixture was worked up by adding 30 mL each of water andethyl acetate. The mixture was transferred to a separatory funnel andthe organic layer was washed with 1N HCl (10 mL), water (30 mL) andbrine (20 mL) and dried over magnesium sulfate. The filtrate afterdrying was evaporated to dryness and crystallized with methyl t-butylether. The resulting compound was >95% pure by HPLC and ¹H NMR analysisconfirmed the esterification at the 2′-hydroxyl of the paclitaxelwithout affecting the 7-hydroxyl thereof. Yield was 98%.

B. Preparation of 2′aldehyde derivative of paclitaxel:

To a solution of 2′-hexenoate of paclitaxel (0.475 g, 0.5 mM) in t-BuOH,acetone and water (2 mL each) were added NMO (0.118 g, 1 mM), NalO₄(0.214 g, 1 mM) and OsO₄ (2.54 mg, 0.01 mM) solution in t-BuOH under anatmosphere of nitrogen. The resulting mixture was stirred at ambienttemperatures for 2 h and at this time, TLC showed completion of thereaction. The mixture was worked up by adding water and ethyl acetate(20 mL each), the organic layer was separated and washed with 1N HCl (10mL), water (10 mL) and brine (10 mL). The organic layer was filteredover magnesium sulfate and sodium hydrosulfite and evaporated todryness. The crude compound was purified by column chromatography usingethyl acetate and heptane, which was characterized by MS and ¹H NMR.Yield: 85%.

C. Preparation of 2′diol derivative of paclitaxel:

As shown in FIG. 4( a), when NalO₄ was not used in the above reaction,the diol was isolated, as follows:

To a solution of the 2′-hexenoate of paclitaxel (9.0 g, 9.47 mM) inacetone (135 mL) was added water (50 mL). To the resulting solution wasadded NMO (2.22 g, 18.94 mM) followed by OsO₄ solution in t-BuOH (48.2mg, 0.19 mM) under an atmosphere of nitrogen and left stirring for 16 h.After confirming completion of the conversion by TLC, diatomaceous earth(15 g) was added to the reaction mixture and filtered. The filtrate wasevaporated free of acetone on the rotavapor followed by extraction withethyl acetate (200 mL) after saturating the aqueous layer with solidNaCl. The resulting organic layer was washed with water (10 mL), 1N HCl(100 mL), water (10 mL) and brine (100 mL) and filtered through MgSO₄.The solvent was evaporated to a residue which was purified by columnchromatography to yield the pure diol in 65% yield. As with thecorresponding diol of the 7-paclitaxel derivative discussed above, theoxidative cleavage of the diol on treatment with NalO₄ provides theterminal aldehyde.

D. Preparation of Transferrin-2′-acyl-hexanal paclitaxel conjugates:

As shown in FIG. 4( b), paclitaxel was first converted to a2′-acyl-hexanal paclitaxel compound through various intermediates. Thechemistry for forming the 2′-acyl-hexanal paclitaxel aldehyde esterderivative is similar to that described above with respect to formingthe 2′-acyl-pentanal paclitaxel derivative, except that 6-heptenoic acidis used in place of the 5-hexenoic acid.

The aldehyde ester is then linked to Transferrin to form aTransferrin-2′-paclitaxel conjugate having conjugation number n asfollows:

The procedure was similar to that described above with respect to thepreparation of a Transferrin-7-paclitaxel conjugate. Mass Spectrometryrevealed that the major Transferrin-2′-paclitaxel conjugate product hada coupling ratio of one paclitaxel molecule per Transferrin (i.e. n=1),although conjugates with two paclitaxel molecules to each Transferrin(i.e. n=2) were also detected. Circular dichroism spectra of Transferrinand the Transferrin-2′-paclitaxel conjugate in the far UV region weredifferent, indicating that the 2′-paclitaxel conjugates might have achanged overall conformation.E. Preparation of Transferrin-2′acyl-nonanal paclitaxel conjugate:

As shown in FIG. 4( c), a Transferrin conjugate of 2′-paclitaxel wasformed by first converting paclitaxel to a 2′-acyl-nonanal paclitaxelaldehyde ester compound through various intermediates. While thechemistry is again similar to that described above with respect to the2′-acyl-pentanal paclitaxel derivative, it should be noted that oleicacid (cis-9-octadecenoic acid: CH₃(CH₂)₇CH═CH(CH₂)₇CO₂H) was used as theacylating agent, as shown in FIG. 4( c). Accordingly, when the chain wascleaved to form the aldehyde, that portion of the chain extending beyondthe 9,10 double bond was removed.

The conjugation of the 2′-acyl-nonanal paclitaxel with Transferrin toform a Transferrin-2′-paclitaxel conjugate of the following formula:

was similar to that described above with respect to theTransferrin-7-paclitaxel conjugate.VI. Amido Derivatives

The present invention also contemplates the formation ofamido-derivatives of amine-bearing compounds. For example, a paclitaxelanalog of the formula:

could be coupled with an appropriate acylating agent, preferably havinga terminal olefin, thereby to form an amido derivative of paclitaxelthat can be converted into an aldehyde linker for use with Transferrinor other carrier molecules/proteins. An exemplary intermediate formed bycoupling the above amine-bearing paclitaxel analog with hexenoic acid isas follows:

Such an intermediate could be converted to the corresponding diol:

and the aldehyde:

according to the processes disclosed above with respect to the7-paclitaxel and 2-paclitaxel derivatives, for example. A Transferrinconjugate of the formula:

is accordingly contemplated. The use of linking compounds having alonger chain length than hexenoic acid is contemplated to address anydifficulties with forming the Transferrin conjugate due to hindrancefrom positioning of the side chain in the concave region around C-13 ofthe hemispherical taxane skeleton. For example, the use of heptenoicacid resulted in the formation of a Transferrin conjugate of theformula:

Here, a protected taxane was first converted to its correspondingamine-hydrochloride salt, as follows:

The formation of such a protected taxane, such as the7-O,3′-N-di-(CBZ)-2′-O-BOM paclitaxel shown in the formula above, isknown in the art and is disclosed, for example, in U.S. Pat. Nos.5,750,737; 5,973,170; 6,133,462; 6,066,749; 6,048,990; 6,136,999; and6,143,902, the teachings of which are incorporated herein by reference.The formation of the amine-hydrochloride salt shown in the reactionabove is described more fully in U.S. patent application Ser. No.09/843,284, the teachings of which are also incorporated herein byreference.

For example, in an exemplary reaction, 5.05 g of7O,3′-N-di-(CBZ)-2′-O-BOM paclitaxel was dissolved in 90.0 mL of THF inan 0.5 L round bottom flask equipped with a magnetic stir bar, to whichwas added 6.01 mL of 3.62M hydrochloric acid (22.08 mmol) and 8.10 g of10% Pd/C 50% wet. The reaction vessel was flushed three times withnitrogen and two times with hydrogen, and the reaction mixture wasstirred vigorously under an atmosphere provided by a hydrogen filledballoon for about one hour at room temperature. This results in theamine-hydrochloride salt shown in the reaction above. It should beappreciated that other mineral acids, as well as organic acids, may beused in place of the hydrochloric acid used in the above process.

Other processes are known for forming the ammonium salts of taxanes. Forexample, the use of trifluoroacetic acid to form the correspondingammonium trifluoroacetate (TFA) salt is disclosed, for example, in U.S.Pat. Nos. 5,675,025; 5,684,175; 5,770,745; 5,939,566; 5,948,919;6,048,990; 6,066,749; 6,072,060; 6,136,999; 6,143,902; 6,262,281; and6,307,088, the teachings of which are incorporated herein by reference.

Once the amine-hydrochloride acid salt was formed, it was then reactedwith heptenoic acid to form the corresponding heptenoate as follows:

This reaction is generally similar to those disclosed above withreference to 7-paclitaxel derivatives or 2′-paclitaxel derivatives,except that it should be appreciated that triethylamine (TEA) is addedto free the amine salt to its corresponding free amine. The resultingheptenoate was then converted to the corresponding 3′-amido-hexanalpaclitaxel as follows:

This reaction is again similar to that described with reference to otherderivatives, above. The resulting aldehyde was then linked withTransferrin using a procedure similar to that described above withrespect to forming the Transferrin-7-paclitaxel conjugates to providethe Transferrin-3′-amido-hexanal paclitaxel conjugate. The circulardichroism spectra of Transferrin and of the Transferrin-3′-amido-hexanalpaclitaxel conjugate were similar, indicating that they had a similaroverall conformation.

It should be appreciated from the foregoing that the corresponding freeamine itself can be used in place of the amine salt. The formation ofthe free amine of taxanes is disclosed, for example, in U.S. Pat. Nos.5,688,977; 5,770,745; 5,939,566; 6,048,990; 6,066,749; 6,072,060;6,107,497; 6,262,281; and 6,307,088, the teachings of which areincorporated herein by reference. Accordingly, the present inventioncontemplates substituting for the amine-hydrochloride salt in theexample above compounds of general formula:

and their analogs and derivatives, where R₃ can be NH₂ or NH₂HA where HAis an organic acid or mineral acid.VII. Generalized Procedures and Compounds

As apparent from the foregoing discussion, the present invention lendsitself to a generalized procedure for forming protein-drug conjugates. Ahydroxyl-bearing or amino-bearing biologically active compound, or ananalog or derivative thereof, of the formula R₁—NH₂ or R₁—OH is firstprovided, which may optionally be protected by one or more protectinggroups on other positions to the extent known in the art. It should alsobe appreciated that the present invention contemplates secondary aminesof the amino-bearing biologically active molecules (i.e. of formulaR₁R₄NH, where R₄ is any appropriate radical as known in the art).

The biologically active compound is reacted with a compound selectedfrom the formulas:

wherein W, X and Y are as described above, thereby to form a compoundhaving the formula:

where Z is —O— when the biologically active compound is of the formulaR₁—OH and Z is —NH— where the biologically active compound is of theformula R₁—NH₂, and W and Y are as above. This compound is oxidized toan aldehyde of the formula:

Alternatively, the corresponding diol of formula:

may be formed as an intermediate that may be cleaved to the aldehyde.The aldehyde is linked with a protein or other carrier molecule havingaccessible amino functionalities, such as a compound of a generalizedformula:

where m is an integer, P is a protein or other carrier molecule, and(NH₂)_(m) are the accessible amino functionalities thereof, thereby toform a conjugate of the formula:

wherein n is the conjugation number of the molecular conjugate, whichreflects the number of molecules of a given drug that are linked to asingle carrier molecule, and which may vary based on the reactionconditions and underlying intermediate compounds used to link a givendrug to a carrier molecule, such as a Transferrin protein.

Carrier molecules contemplated for use in the present invention includeproteins such as Transferrin, the receptor ligand peptides recognized bySafavy, or other proteins, antibodies, lectins or other substances thatmay become attached to the surface of a cell.

It should also be appreciated that this generalized procedurecontemplates the formation of various intermediate compounds, such asthe olefins, diols and aldehydes of general formula:

wherein R₁, Z and Y are as above and R₂ is —CH═CH(W), —CH(OH)CH(OH)W, or—C(O)H, where W is as above. As appropriate, R₁ may include one or moreprotecting groups, such as TBDMS, TROC, BOM, benzyl, TES or EE in thecase of paclitaxel, for example. In such case, the method may includesteps of protecting and deprotecting R₁, as appropriate, with one ormore protecting groups. For example, paclitaxel may be protected at the2′ site with TBDMS, TROC, BOM, benzyl, TES or EE, or the like, prior tothe step of coupling it with the acylating agent, and may thereafter bedeprotected at 2′ after the step of converting the compound to itscorresponding aldehyde.VIII. Analysis/Characterization

Transferrin-drug conjugate products formed according to theabove-described methods were characterized by mass spectrometry andFPLC. UV Spectra were collected from 200 nm to 800 nm. Theconcentrations of the Transferrin/Transferrin conjugates were 0.5 mg/ml.Samples were prepared in PBS (pH=7.4) buffer. Circular dichroismexperiments were carried out by collecting spectra from 240 nm to 190 nmwith a cylindrical quartz cell of path length 1 mm. The concentrationsof the Transferrin/Transferrin conjugates were 1 mg/ml. Samples wereprepared in H₂O or PBS (pH=7.4) buffer.

The Transferrin, rhodamine123 and Transferrin-rhodamine123 conjugatewere evaluated using a fluorescence spectrofluorophotometer. TheTransferrin (0.25 mg/ml), rhodamine123 (10 ng/ml) andTransferrin-rhodamine123 conjugate (0.25 mg/ml) were dissolved inphosphate buffer (pH 7.0). The excitation wavelength was 280 nm forTransferrin and Transferrin-rhodamine123 conjugate, 500 nm forrhodamine123, and emission spectra were recorded in the range of 300 to700 nm.

The gel-electrophoresis technique of Laemmli (“Cleavage of StructuralProteins During the Assembly of the Head of Bacteriophage T4”, Nature,227, 680–685 (1970)) was used to assess the purity of the Transferrinconjugates. Sodium dodecyl sulphate (SDS) polyacrylamide gelelectrophoresis of the conjugate column fractions were performed with avertical slab gel composed of 12% acrylamide (NuPAGE electrophoresissystem, NOVEX®) and run in a E1900-XCELL™ Mini Cell (Novex, San Diego,Calif.) apparatus. Samples were prepared and loaded on the gel afterheating at 100° C. for 3 min. The Transferrin conjugates (20 μl) wereloaded on the gel at approximately 0.03 mg/ml protein. When theelectrophoresis was completed, the gels were stained for 30 min withCoomassie blue stain solution and then destained for 5–12 h. In the caseof the Transferrin-7-paclitaxel conjugate, samples were analyzed inpresence and absence of 2-mercaptoenthanol (1 μl, a reducing reagent).

The standard curve of pacilitaxel by HPLC was obtained by injection ofknown quantities of paclitaxel and plotting the peak area vsconcentration of paclitaxel. The sample was analyzed by analyticalreversed-phase HPLC using a C-4 column (5 μm, 300 Å, 25 cm×4.6 mm i.d.,flow rate 1 ml/min) eluting gradient of solvent A, 80% H₂O: 20% ACN:0.1% TFA, and solvent B, 80% ACN: 20% H₂O: 0.1% TFA. The total HPLCanalysis run was 24 min. The gradient method used for the analyticalHPLC was started from solvent B from 0% to 100% over 20 min, followed byelution at 100% of solvent B for 2 min. Finally, a gradient change backto 0% solvent B was done over 2 min.

Stock solution (1 mg/ml) of paclitaxel was prepared by dissolvingpaclitaxel in EtOAc. The final concentrations of paclitaxel were 25, 50,75, 100 μg/ml. For each concentration, sample was injected into HPLC induplicate and the standard curve of pacilitaxel was obtained by plottingthe averaged peak area vs concentration of paclitaxel.

The coupling ratio of the Transferrin-7-paclitaxel conjugate wasmeasured after an acid hydrolysis of the conjugate followed bymeasurement of the paclitaxel by HPLC. 1 mg Transferrin-paclitaxel wasdissolved in 0.4 ml PBS buffer, pH was adjusted to 4 by adding aceticacid and the reaction mixture was stirred at room temperature for 10min. Paclitaxel was isolated by adding 0.2 ml EtOAc into reactionmixture and vortexing for 2 min. The turbid mixture was then centrifugedat 1000 g for 10 min at 4° C. and the clear supernatant 20 μl thatcontained paclitaxel was injected into HPLC. According to the standardcurve of paclitaxel, there was a coupling ratio of 3 paclitaxel perTransferrin.

IX. Stability of the Transferrin Conjugates

A. Thermal Stability

Stock solutions of Transferrin and Transferrin conjugates were preparedby dissolving Transferrin and Transferrin conjugates (1 mg/ml) in PBSbuffer (pH 7.0, 0.05 mol). Aliquoted 0.1 ml into sealed tubes andincubated at 37° C. At appropriate time intervals, aliquots were removedin triplicate, frozen immediately in dry ice and stored at −70° C. untilanalysis by CD and SDS-PAGE Electrophoresis. Immediately prior toanalysis the appropriate sample was fast-thawed.

B. pH-Dependent Stability

Stock solutions of Transferrin and Transferrin conjugates were preparedby dissolving Transferrin and Transferrin conjugates (1 mg/ml) in H₂O.The stock solutions were further diluted in different pH buffer (0.05mol) and the final concentration was 0.1 mg/ml. Samples were incubatedat room temperature for 2 h, or at 37° C. for 2 h analysis by CD,Fluorescence and UV Spectroscopy.

X. Cytotoxicity Data

The growth inhibitory potential of the Transferrin-3-cholesterolconjugate, the Transferrin-rhodamine123 conjugate and theTransferrin-7-paclitaxel conjugate with cultured mammalian cells wasinvestigated. As discussed below, the Transferrin conjugates ofrhodamine123 and cholesterol did not demonstrate significant adverseeffect against either tumor or normal cells. This demonstrates thatconjugation of substances to Transferrin by itself is not sufficient tocause effect on cell growth. However, the conjugate of paclitaxel, acompound exhibiting efficacy against cancer, showed promise in targetingcancer cells while not adversely affecting normal cells. Thus, thepresent invention suggests a promising route to specifically targetcancer cells with compounds that exhibit efficacy in cancer treatment,with the potential of not harming normal cells at optimal doses.

The cell lines selected were KB (human epidermoid carcinoma in themouth, ATCC #CCL-17), Lu-1 (human lung cancer cell lines, obtained fromthe Department of Surgical Oncology, University of Illinois, College ofMedicine), and hTERT (telomerase-immortalized normal epithelial cellline, Clontech #C4000-1). Various doses of the compounds were evaluatedunder two treatment protocols. In Protocol A, cultures of each of theKB, Lu and hTERT cells were treated with various doses of the threeconjugate compounds from the day of culture onward. In Protocol B,cultures of each of the KB, Lu and hTERT cells were grown to confluence(9 days), and then treated with various doses of theTransferrin-7-paclitaxel conjugate for the duration of the experiment.

KB was cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum(FBS), 100 units/ml penicillin G, 100 μg/ml streptomycin sulfate, 0.25μg/ml amphotericin B (Fungizone) (PSF) (GIBCO) and 1% non-essentialamino acids (NAA) (Sigma). Lu was maintained in MEME (GIBCO)supplemented with 10% FBS, PSF, and 1% NAA. hTERT-RPE1 was maintained inDMEM/F-12 (GIBCO) supplemented with 10% FBS+PSF. All cell lines werecultured at 37° C. in 100% humidity with a 5% CO₂ atmosphere in air.

The overall procedures were those as described by Skehan et al., Newcolorimetric cytotoxicity assay for anticancer-drug screening, J. Natl.Cancer Inst. 82: 1107–1112, 1990, and Likhitwitayawuid et al., Cytotoxicand antimalarial bisbenzylisoquinoline alkaloids from Stephania erecta.J. Nat. Prod. 56: 30–38, 1993. Cells were typically grown to 60–70%confluence, the medium was changed, and the cells were used for testprocedures one day later. Test samples were initially dissolved insterilized PBS. Serial dilutions were performed using PBS as thesolvent, and 10 μl were added to the three wells. PBS (10 μl) was addedto control groups. After the plates were prepared, cells were removedfrom the tissue culture flasks by treatment with trypsin, enumerated,and diluted with fresh media. KB (3×10⁴ cells/ml), Lu (5×10⁴ cell/ml)and hTERT-RPE1 (4×10⁴ cells/ml) cells (in 190 μl of media) were added tothe 96-well plates. The plates were incubated at 37° C. in 5% CO₂, thecells fixed by addition of 100 μl of cold 20% trichloroacetic acid andincubated at 4° C. for 30 min. The plates were washed with tap water(3×) and dried overnight. The fixed cells were stained 30 min by theaddition of 100 μl of 0.4% sulforhodamine B (w/v) dissolved in 1% aceticacid. The plates were washed with 1% acetic acid (3×) and allowed todry. The bound dye was solubilized by the addition of 10 mM unbufferedTris base, pH 10 (200 μl/well). The plates were placed on a shaker for 5minutes, and the absorption was determined at 515 nm using an ELISAplate reader. In each case, a zero-day control was performed by addingan equivalent number of cells to several wells of the 96-well plates andincubating at 37° C. for a period of 30 min. The cells were then fixedwith tichloroacetic acid and processed as described above.

The first series of studies were conducted with theTransferrin-3-cholesterol conjugate, the Transferrin-rhodamine123conjugate and the Transferrin-7-paclitaxel conjugate following ProtocolA, in which various concentrations of test compounds ranging from62.5–250 μg/ml were added on the day of plating, and media werereplenished every 3 days, maintaining the same concentrations of thetest compounds. As shown by plotting optical density versus time, theTransferrin-3-cholesterol conjugate and the Transferrin-rhodamine123conjugate were not active with any of the cell lines (FIGS. 5, 6, 8, 9,11, 12). On the other hand, complete inhibition of cell growth wasobserved with all of the concentrations of the Transferrin-7-paclitaxelconjugate that were tested (FIGS. 7, 10, 13).

Therefore, the experiment was repeated with the Transferrin-7-paclitaxelconjugate with lower concentrations. As illustrated in FIG. 14, usingProtocol A and KB cells, complete growth inhibition was observed withconcentrations of 5 or 50 μg/ml, but no effect on growth was observed ata concentration of 0.5 μg/ml. Similar responses were observed with Luand hTERT cells, as illustrated in FIGS. 15 and 16, respectively.

A similar profile was observed with KB cells following Protocol B, inwhich cells were grown until the 9^(th) day without changing the media,various concentrations of test compounds were added on the 9^(th) day,and compounds and media were then replenished every 3 days. Atconcentrations of 50 or 5 μg/ml, significant reduction in cell numberwas observed, but at 0.5 μg/ml, cell number paralleled that of thecontrol (FIG. 17).

Lu cells were somewhat more resistant. At 50 μg/ml, a significantreduction in cell number was observed. This was diminished at aconcentration of 5 μg/ml, and negated at a concentration of 0.5 μg/ml(FIG. 18).

The weakest effect was observed with hTERT cells. Slight increases incell growth were observed in the control cultures and cultures treatedwith 0.5 or 5 μg/ml of the Transferrin-7-paclitaxel conjugate. Thisgrowth was diminished when the cells were treated with 50 μg/ml, but onday 16, the cell number was still approximately the same as on day 9(FIG. 19). These results suggest that the Transferrin-7-paclitaxelconjugate is primarily targeting cancer cells (KB and Lu) while notsignificantly affecting normal cells (hTERT), indicating that theTransferrin-7-paclitaxel conjugate and other protein-drug conjugatesaccording to the present invention may provide a promising route tocancer treatment.

Accordingly, the present invention has been described with some degreeof particularity directed to the exemplary embodiments of the presentinvention. It should be appreciated, though, that the present inventionis defined by the following claims construed in light of the prior artso that modifications or changes may be made to the exemplaryembodiments of the present invention without departing from theinventive concepts contained herein.

1. A compound having the formula:

wherein (a) R₁ is selected from: (1) a de-hydroxyl moiety of (i) ahydroxyl-bearing biologically active molecule and analogs andderivatives thereof of formula R₁—OH, and (2) a de-amino moiety of (i)an amino-bearing biologically active molecule and analogs andderivatives thereof of formula R₁—NH₂ or a salt or secondary aminethereof; (b) where Z is —O— when R₁ is said de-hydroxyl moiety and Z is—NH— when R₁ is said de-amino moiety; (c) R₂ is selected from—CH(OH)CH(OH)W, and —C(O)H; (d) W is selected from: (1) H, (2) astraight or branched alkyl having 1 to 20 carbons optionally substitutedwith one or more phenyl, (3) a cycloalkyl optionally substituted withone or more alkyl or phenyl, and (4) an aromatic group optionallysubstituted with one or more alkyl or electron-withdrawing orelectron-donating groups; and (e) Y is selected from: (1) a straight orbranched alkyl having 1 to 20 carbons optionally substituted with one ormore phenyl, (2) a cycloalkyl optionally substituted with one or morealkyl or phenyl, and (3) an aromatic group optionally substituted withone or more alkyl or electron-withdrawing or electron-donating groups.2. A compound according to claim 1 wherein R₂ is selected from—CH(OH)CH₂(OH) and —C(O)H.
 3. A compound according to claim 1 whereinsaid biologically active molecule is a drug useful in cancer therapy. 4.A compound according to claim 3 wherein said drug is a cancertherapeutic drug.
 5. A compound according to claim 3 wherein said drugis selected from taxanes, camptothecins, epothilones, cucurbitacins,quassinoids, anthracyclines, and their analogs and derivatives.
 6. Acompound having the formula:

wherein (a) R₁ is a 2′-dehydroxyl paclitaxel moiety; (b) Z is —O—; (c)R₂ is selected from —CH═CH(W), —CH(OH)CH(OH)W, and —C(O)H; (d) W isselected from: (1) H, (2) a straight or branched alkyl having 1 to 20carbons optionally substituted with one or more phenyl, (3) a cycloalkyloptionally substituted with one or more alkyl or phenyl, and (4) anaromatic group optionally substituted with one or more alkyl orelectron-withdrawing or electron-donating groups; (e) and Y is—(CH₂)_(r)— where r is an integer from 4 to
 7. 7. A compound having theformula:

wherein (a) R₁ is selected from: (1) a de-hydroxyl moiety of (i) ahydroxyl-bearing biologically active molecule and analogs andderivatives thereof of formula R₁—OH provided that R₁ is not a taxane ora 3-dehydroxyl cholesterol, and (2) a de-amino moiety of (i) anamino-bearing biologically active molecule and analogs and derivativesthereof of formula R₁—NH₂ or a salt or secondary amine thereof; (b)where Z is —O— when R₁ is said de-hydroxyl moiety and Z is —NH— when R₁is said de-amino moiety; (c) R₂ is selected from —CH═CH₂,—CH(OH)CH₂(OH), and —C(O)H; (d) W is selected from: (1) H, (2) astraight or branched alkyl having 1 to 20 carbons optionally substitutedwith one or more phenyl, (3) a cycloalkyl optionally substituted withone or more alkyl or phenyl, and (4) an aromatic group optionallysubstituted with one or more alkyl or electron-withdrawing orelectron-donating groups; and (e) Y is selected from: (1) a straight orbranched alkyl having 1 to 20 carbons optionally substituted with one ormore phenyl, (2) a cycloalkyl optionally substituted with one or morealkyl or phenyl, and (3) an aromatic group optionally substituted withone or more alkyl or electron-withdrawing or electron-donating groups.